The wet surface of the eye is crucial for light refraction. Drying diseases and pathogen invasion often occur on this exposed surface. Mucins of two types[unreadable]secreted and membrane associated[unreadable]are hypothesized to be vital for maintenance of the wet surface. Membrane associated mucins present in a dense layer on the apical cells of the cornea! epithelium at the tear film interface are also hypothesized to be a barrier to pathogen and large molecule penetrance. Building on the characterizations of membrane associated mucins of the ocular surface and on the systems developed for their study in our previous funding period, we propose to investigate the function of this class of mucins at the surface of the eye in six aims. Aim I: We will determine if membrane-associated mucins are involved in surface membrane microplicae formation on cornea! surfaces. Aim II: We will determine if membrane-associated mucins give rise to the changes on surface cells indicative of apical cell differentiation of the ocular surface epithelia. Aim III: We will determine if membrane-associated mucins function as disadhesives, preventing cell or bacterial adhesion. In Aims Mil, the studies will be done using a human cornea! limbal epithelial cell line, along with siRNA technology, field emission immuno-scanning electron microscopy, and real time PCR. Data from the cell line will be correlated to that of wild type and mice null for membrane associated mucins. Aim IV: We will determine if membrane associated mucins form a barrier to penetrance of large molecules and pathogens along the glycocalyx present on the tips of the microplicae of the corneal epithelium, and if the secreted goblet cell mucin MUC5AC is partitioned outside that barrier. Rapid freeze, freeze substitution followed by immunoelectron microscopy will be used to localize pathogens and MUC5AC in relation to membrane associated mucins. Aim V: We will determine if membrane associated mucins protecting the eye from fluid loss and dry eye, using mice null for membrane associated mucins housed in a controlled environment chamber which reliably produces decreased tear volume in mice. Aim VI: We will determine if the alteration of MUC16 seen previously in dry eye is a result of a decrease in mucin gene expression, shedding rate, and/or post-translational modification, and if the extent of these alterations correlates with severity of dry eye.